Abstract 16433: TGF-beta Regulates Cytosolic Mir-21 Maturing in Mouse and Human Cardiac Fibroblasts Under Pressure Overload
Transforming growth factor β (TGFβ) promotes LV remodeling under pressure overload. TGFβ induces in this situation miR21 overexpression which contributes to the LV profibrotic events in mice and patients. In cultured cells, the interaction of TGFβ signaling Smad effectors with DROSHA induces post-transcriptional processing of primary miR21 into precursor miR21 (pre-miR21) in the nucleus.
Hypothesis: TGFβ regulates the cytosolic, DICER-dependent, processing of pre-miR21 to mature miR21 in cardiac fibroblasts under experimental and clinical pressure overload stress.
Methods and Results: The subjects of the study were mice undergoing transverse aortic constriction (TAC; n=15) and patients with aortic stenosis (AS; n=67) and controls (n=30). Studies in cultured cells were performed in NIH-3T3 and primary LV fibroblasts. mRNA, miRNA (PCR) and protein (western blot) expressions were determined in LV samples from patients and mice. Mature miR21 expression was detected in NIH-3T3 cells and primary cardiac fibroblasts by in situ hybridization. Fibroblasts responded upon TGFβ stimulation overexpressing miR21, while silencing Smad2/3 with siRNA prevented pre-miR21 processing to mature miR21. Confocal immunofluorescence and coimmunoprecipitation demonstrated DICER-p-Smad2/3 interactions in fibroblasts. In mice TAC produced LV overexpression of DICER [gene (Sham: 5.9±0.3 vs TAC: 12.6±0.7***) and protein]. DICER mRNA levels correlated with Smad2 (R = 0.79***), Smad3 (R = 0.54**) and pre-miR21 (R = 0.62**). Multiple linear regression depicted TGFβ, Smad2 and Smad3 as significant predictors of DICER expression in the LV under biomechanical stress (adjusted R2: 0.70***). Pre-miR21 was identified in the immunoprecipitated p-Smad2/3-DICER protein complexes. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with Smad2 (R=0.40 ***), Smad3 (R=0.51***) and mir21 (R=0.33**).
Conclusion: Smad2 and Smad3 interact with DICER to promote the cytosolic processing of pre-miR21 to mature miR21. This new TGFβ-dependent regulatory mechanism is involved in miR21 overexpression in cultured fibroblasts and in the pressure overloaded LV in mice and patients (ISCIII PI12/00999).
- © 2013 by American Heart Association, Inc.